Programmed cell death (apoptosis) is induced concomitant with differentiation in skeletal myoblasts and other systems. In skeletal myoblasts, these two processes result in mutually exclusive physiologically important endpoints. However, the coordinate regulation of these two processes is not understood. Dissecting this coordinate regulation could enable selective manipulation relevant to the amelioration of disease states associated with muscle degeneration and to the effectiveness of any treatment utilizing skeletal myoblast transfer. The long-term goal of this laboratory, therefore, is to elucidate the molecular mechanism(s) responsible for this coordinate regulation. [unreadable] [unreadable] Guided by our preliminary data, the short-term goal of this proposal is to test the hypothesis that MyoD is both sufficient and necessary to induce mitochondrial disruption and apoptosis by affecting one or more of the three pro-apoptotic pathways previously identified by this laboratory to play a role in the apoptotic process in skeletal myoblasts. This hypothesis will be tested by pursuing the following specific aims. [unreadable] [unreadable] Aim 1. Examine the contribution of MyoD to the increased expression of PUMA and to the increased signaling through the TRAIL/DR5/FADD/FLIP/tBid pathway: We will activate ectopically expressed MyoD in 10T1/2 fibroblasts and utilize RNAi to selectively suppress the expression of endogenous MyoD in 23A2 and C2C12 established myoblasts [and in primary skeletal myoblasts]. We will then examine the effect of these manipulations on these two signaling pathways previously determined to play a role during skeletal myoblast apoptosis, and on mitochondrial disruption and apoptosis. We will also compare this apoptotic process in skeletal myoblasts isolated from wild-type and MyoD null mice. [unreadable] [unreadable] Aim 2. Examine the contribution of MyoD to the activation of serine proteases and identification of the serine protease(s) that function prior to the release of cytochrome C during apoptosis: Using FLISP (fluorescent inhibitor of serine proteases) to selectively and covalently label in situ active serine proteases, we have determined that at least 7 serine proteases are specifically activated in apoptotic myoblasts. We will determine the effect of activation of MyoD in fibroblasts and silencing of MyoD in established and primary myoblasts on activation of these proteases. Further, we will compare the activation of these proteases in skeletal myoblasts isolated from wild-type and MyoD null mice. Finally, labeled active serine proteases will be isolated and analyzed by capillary column HPLC-tandem mass spectrometry. Once identification is confirmed, the role of these specific protease(s) in mitochondrial disruption and apoptosis will be examined. PUBLIC HEALTH RELEVANCE. These studies will dissect a new biological role for MyoD and should identify the first serine protease to function prior to mitochondrial disruption and a potentially novel serine protease(s) relevant to apoptosis in other systems. Furthermore, these findings could be significant in the effort to ameliorate the myoblast apoptosis that contributes pathologically to a variety of diseases and in the study of myoblast transfer as a therapeutic approach. [unreadable] [unreadable] [unreadable] [unreadable]